CN115109580A - Polystyrene AIE dye fluorescence encoding microsphere material with carboxyl coated on outer layer, and preparation method and application thereof - Google Patents
Polystyrene AIE dye fluorescence encoding microsphere material with carboxyl coated on outer layer, and preparation method and application thereof Download PDFInfo
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- CN115109580A CN115109580A CN202210511940.1A CN202210511940A CN115109580A CN 115109580 A CN115109580 A CN 115109580A CN 202210511940 A CN202210511940 A CN 202210511940A CN 115109580 A CN115109580 A CN 115109580A
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- 239000004005 microsphere Substances 0.000 title claims abstract description 93
- 239000004793 Polystyrene Substances 0.000 title claims abstract description 57
- 229920002223 polystyrene Polymers 0.000 title claims abstract description 57
- 239000000463 material Substances 0.000 title claims abstract description 36
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 18
- 238000012674 dispersion polymerization Methods 0.000 claims abstract description 13
- 238000001514 detection method Methods 0.000 claims abstract description 8
- 239000007791 liquid phase Substances 0.000 claims abstract description 8
- 230000009145 protein modification Effects 0.000 claims abstract description 6
- 239000011259 mixed solution Substances 0.000 claims description 57
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 43
- 239000000047 product Substances 0.000 claims description 38
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 36
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 36
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims description 32
- 239000000243 solution Substances 0.000 claims description 29
- 238000006243 chemical reaction Methods 0.000 claims description 26
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 18
- 239000000178 monomer Substances 0.000 claims description 15
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 14
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 14
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 14
- 238000001132 ultrasonic dispersion Methods 0.000 claims description 14
- 229920004894 Triton X-305 Polymers 0.000 claims description 13
- 238000003756 stirring Methods 0.000 claims description 13
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 12
- 238000004140 cleaning Methods 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 12
- FRPZMMHWLSIFAZ-UHFFFAOYSA-N 10-undecenoic acid Chemical compound OC(=O)CCCCCCCCC=C FRPZMMHWLSIFAZ-UHFFFAOYSA-N 0.000 claims description 11
- KHAVLLBUVKBTBG-UHFFFAOYSA-N caproleic acid Natural products OC(=O)CCCCCCCC=C KHAVLLBUVKBTBG-UHFFFAOYSA-N 0.000 claims description 11
- 229960002703 undecylenic acid Drugs 0.000 claims description 11
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 10
- NFHFRUOZVGFOOS-UHFFFAOYSA-N palladium;triphenylphosphane Chemical compound [Pd].C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 NFHFRUOZVGFOOS-UHFFFAOYSA-N 0.000 claims description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- 235000019441 ethanol Nutrition 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 238000010992 reflux Methods 0.000 claims description 9
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 claims description 8
- OZAIFHULBGXAKX-VAWYXSNFSA-N AIBN Substances N#CC(C)(C)\N=N\C(C)(C)C#N OZAIFHULBGXAKX-VAWYXSNFSA-N 0.000 claims description 8
- 239000007795 chemical reaction product Substances 0.000 claims description 8
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 claims description 8
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 6
- 239000003513 alkali Substances 0.000 claims description 6
- 238000004440 column chromatography Methods 0.000 claims description 6
- 239000006185 dispersion Substances 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 6
- 239000003208 petroleum Substances 0.000 claims description 6
- 239000002243 precursor Substances 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- KEOLYBMGRQYQTN-UHFFFAOYSA-N (4-bromophenyl)-phenylmethanone Chemical compound C1=CC(Br)=CC=C1C(=O)C1=CC=CC=C1 KEOLYBMGRQYQTN-UHFFFAOYSA-N 0.000 claims description 5
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims description 5
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- XJDNKRIXUMDJCW-UHFFFAOYSA-J titanium tetrachloride Chemical compound Cl[Ti](Cl)(Cl)Cl XJDNKRIXUMDJCW-UHFFFAOYSA-J 0.000 claims description 5
- 125000000524 functional group Chemical group 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- 239000012467 final product Substances 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 150000002576 ketones Chemical class 0.000 claims description 3
- 239000012046 mixed solvent Substances 0.000 claims description 3
- 238000006116 polymerization reaction Methods 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 2
- 239000004327 boric acid Substances 0.000 claims description 2
- 238000009833 condensation Methods 0.000 claims description 2
- 230000005494 condensation Effects 0.000 claims description 2
- 238000001308 synthesis method Methods 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 abstract description 7
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- 238000003018 immunoassay Methods 0.000 abstract description 3
- 102000039446 nucleic acids Human genes 0.000 abstract description 3
- 108020004707 nucleic acids Proteins 0.000 abstract description 3
- 150000007523 nucleic acids Chemical class 0.000 abstract description 3
- 229920006037 cross link polymer Polymers 0.000 abstract description 2
- 238000003786 synthesis reaction Methods 0.000 abstract description 2
- 102000004169 proteins and genes Human genes 0.000 abstract 1
- 108090000623 proteins and genes Proteins 0.000 abstract 1
- 239000000975 dye Substances 0.000 description 38
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- 230000002776 aggregation Effects 0.000 description 4
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- 238000012986 modification Methods 0.000 description 4
- 238000010791 quenching Methods 0.000 description 3
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- 238000005481 NMR spectroscopy Methods 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 238000005054 agglomeration Methods 0.000 description 2
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- 239000007850 fluorescent dye Substances 0.000 description 2
- HXITXNWTGFUOAU-UHFFFAOYSA-N phenylboronic acid Chemical compound OB(O)C1=CC=CC=C1 HXITXNWTGFUOAU-UHFFFAOYSA-N 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
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- 238000000695 excitation spectrum Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 239000002077 nanosphere Substances 0.000 description 1
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- 239000003960 organic solvent Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
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- 239000007787 solid Substances 0.000 description 1
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
- C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
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- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F112/00—Homopolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring
- C08F112/02—Monomers containing only one unsaturated aliphatic radical
- C08F112/04—Monomers containing only one unsaturated aliphatic radical containing one ring
- C08F112/06—Hydrocarbons
- C08F112/08—Styrene
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
The invention discloses a polystyrene AIE dye fluorescence coding microsphere material with an outer layer coated with carboxyl, a preparation method and application thereof. The AIE dye fluorescence coding microsphere material is used for a liquid phase chip detection system for carrying out protein modification. The invention uses simple carboxyl cross-linked polymer to encapsulate and coat the microspheres, improves the stability of the microspheres, realizes the fluorescent coding of the microspheres by a two-step dispersion polymerization method, has simple synthesis process, can keep excellent aggregate fluorescence, and can modify subsequent protein by carboxyl on the surface; the invention develops a novel coding vector for a liquid-phase chip, and provides a novel method for immunoassay, nucleic acid detection analysis and disease diagnosis auxiliary analysis.
Description
Technical Field
The invention relates to a novel AIE fluorescent material, a preparation process and application thereof, which are applied to the technical field of fluorescence detection analysis and biological materials.
Background
The high-throughput suspension array technology has the advantages of simple operation, high acquisition speed, small sample amount and the like, and is widely applied to the fields of immunoassay, nucleic acid detection, disease diagnosis, drug screening and the like, wherein the coding microspheres are key materials for distinguishing different signals in multiplex analysis. However, commercially available fluorescent coded spheres are limited and extremely expensive. The method has important significance for meeting the practical application and developing a fluorescent microsphere coding strategy which is more reliable, more cost-effective and larger in coding capacity.
Organic fluorophores and quantum dots are the most commonly used materials for the preparation of fluorescently encoded microspheres. The quantum dots have wide excitation spectrum and narrow emission spectrum, but the quantum dots are difficult to synthesize, expensive and easy to agglomerate and quench under the influence of environment, so that the application of the quantum dots is limited. To date, most of the liquid phase chip systems reported in the literature employ organic dye-encoded microspheres. The dye-encoded microspheres can be prepared by methods such as copolymerization, covalent connection, adsorption, swelling and the like of dyes. The solvent swelling method is to incorporate dye molecules into microspheres by swelling with an organic solvent, but the encoding step is time-consuming and laborious. Besides optical coding of the microspheres, surface modification of functional groups is also important for preparation of the microspheres, and silicon dioxide is a good material for coating the microspheres.
Quenching of agglomeration of quantum dots is an annoying problem, although many methods have been developed to counteract it with little success, and contrary to quenching of agglomeration of quantum dots is AIE materials, which typically have a propeller or rotor-like structure that is detrimental to the formation of pi-pi stacking in the aggregated state, thereby reducing non-radiative decay in the aggregated state. Although AIE materials have been applied to a wide variety of biomaterials, the data in the binding of nanospheres is also somewhat weak and does not support the acquisition of more stable, richer, easily tracked and analyzed AIE fluorescent materials.
Disclosure of Invention
In order to solve the problems of the prior art, the invention aims to overcome the defects in the prior art, and provides a polystyrene AIE dye fluorescence coding microsphere material with an outer layer coated with carboxyl, a preparation method and application thereof, and relates to a novel PAIE dye coated undecylenic acid fluorescence coding microsphere.
In order to achieve the purpose of the invention, the invention adopts the following technical scheme:
a polystyrene AIE dye fluorescence coding microsphere material with an outer layer coated with carboxyl is prepared by a two-step synthesis method to prepare an AIE monomer, then the AIE monomer is subjected to two-step dispersion polymerization reaction, and the surface of a polymerization product is coated with undecylenic acid, so that a carboxyl functional group is modified on the surface of the microsphere, and finally the polystyrene AIE dye fluorescence coding microsphere material with the surface containing the carboxyl is obtained.
Preferably, the polystyrene AIE dye fluorescent coding microsphere of the polystyrene AIE dye fluorescent coding microsphere material coated with carboxyl groups on the outer layer is a microsphere with the average particle size of 3 +/-0.1 mu m.
The invention relates to a preparation method of a polystyrene AIE dye fluorescence coding microsphere material with carboxyl coated on the outer layer, which comprises the following steps:
a. preparation of green AIE dye monomer material:
a-1, adding zinc powder and 20mL of dry Tetrahydrofuran (THF) into a three-neck flask with the volume of 250mL, slowly dropwise adding titanium tetrachloride after ice bath, then returning to room temperature, keeping for at least 0.5h, carrying out condensation reflux for at least 2 hours, then adding pyridine, and stirring for at least 10 minutes to obtain a first mixed solution;
a-2, adding 4' -bromobenzophenone and tetraethyl michigan ketone into 20mL of tetrahydrofuran, uniformly stirring to obtain a second mixed solution, slowly dropwise adding the second mixed solution into the first mixed solution prepared in the step a-1, and then condensing, refluxing and standing overnight for reaction;
a-3, stopping the reaction in the step a-2 by taking a potassium carbonate solution with the mass percent not less than 10%, extracting a product solution by using Dichloromethane (DCM), then performing alkali washing on the collected extract, drying by using anhydrous sodium sulfate, and finally performing column chromatography by using a petroleum ether/ethyl acetate mixed solvent with the volume ratio of 15:1 as an eluent to obtain a product A;
a-4, adding the product A prepared in the step a-3, the tetravinylbenzene boric acid and the tetrakis (triphenylphosphine) palladium into a three-neck flask with the volume of 100mL, adding 10mL of tetrahydrofuran and a potassium carbonate solution with the mass percent not less than 10%, refluxing overnight, and reacting to obtain a third mixed solution;
a-5, extracting the third mixed solution prepared in the step a-4 by using Dichloromethane (DCM), then carrying out alkali washing on the collected extract, drying by using anhydrous sodium sulfate, and finally carrying out column chromatography by using petroleum ether as an eluent to obtain a final product B, namely the green AIE dye monomer material;
b. preparing the polystyrene AIE dye fluorescent coding microspheres by dispersion polymerization:
b-1, dissolving polyvinylpyrrolidone (PVP) by using absolute ethyl alcohol as a solvent, adding triton X305(TritonX305), styrene and AIBN, transferring the mixed solution into a 250mL three-necked bottle, performing ultrasonic dispersion, and introducing N 2 At least 30min, and carrying out primary reaction at the temperature of not less than 70 ℃ for at least 1 h;
b-2, adding styrene, absolute ethyl alcohol and 5-20mg of product B into a beaker, performing ultrasonic dispersion, and introducing N 2 Preheating at the temperature of not less than 70 ℃ for at least 30min to obtain a fourth mixed solution, adding the obtained fourth mixed solution into a three-necked bottle, mixing the fourth mixed solution with the primary reaction product solution in the step b-1, and continuously reacting for at least 24h until the reaction is finished;
b-3, after the reaction in the step b-2 is finished, centrifuging the product solution, and centrifuging the obtained milky white liquid for at least 3min at a rotating speed of not less than 2000 r/min;
b-4, after centrifugal treatment in the step b-3, ultrasonically cleaning the product for at least 3 times by using ethanol, and then drying overnight to obtain the polystyrene AIE dye fluorescent coding microsphere;
c. preparing poly (undecylenic acid) coated polystyrene AIE dye fluorescent coding microspheres:
c-1, dispersing 10mg of polystyrene AIE dye microspheres in 10mL of SDS solution with the mass percent of not less than 0.25 wt%, and performing ultrasonic dispersion for at least 10min to obtain a polystyrene AIE dye microsphere dispersion solution;
c-2, sequentially adding 12.5mg KPS, a mixed solution of DVB and methanol with a volume ratio of 60uL being 1:100 and a methanol solution of undecylenic acid with a volume ratio of 100uL being 1:100 into the polystyrene AIE dye microsphere dispersion liquid prepared in the step c-1, and performing ultrasonic dispersion on the mixed solution for at least 10min to obtain a precursor mixed solution;
and c-3, placing the precursor mixed solution prepared in the step c-2 into a three-neck flask with the volume of 25mL, stirring for at least 10min, gradually heating to a temperature of not less than 70 ℃, reacting for at least 8 hours, then centrifugally cleaning the product solution with water for at least three times, and cleaning the product with ethanol for at least three times to finally obtain the polystyrene AIE dye fluorescent coding microsphere with the carboxyl coated on the outer layer.
Preferably, in the step a-1, the mixing ratio of zinc powder, tetrahydrofuran, titanium tetrachloride and pyridine is 0.3 g: 20mL of: 0.36 ml: 0.05 mL.
Preferably, in the step a-2, the mixing ratio of tetrahydrofuran, 4' -bromobenzophenone and tetraethyl michelson is 20 mL: 0.2 g: 0.3 g.
Preferably, in the step a-2, the volume ratio of the second mixed solution to the first mixed solution is 20 mL: 20 mL.
Preferably, in the step a-4, the product a, the tetravinylbenzeneboronic acid and the tetrakis (triphenylphosphine) palladium are mixed in a ratio of 0.2 g: 0.5 g: 20 mg; the adding amount of the potassium carbonate solution with the mass percent not less than 10 percent is not less than 1 mL.
Preferably, in the step b-1, the mass ratio of the absolute ethyl alcohol to the polyvinylpyrrolidone to the triton X305 to the styrene to the AIBN is 18.75:1:0.35:6.25: 0.25.
Preferably, in the step B-2, the mixing ratio of the styrene, the absolute ethyl alcohol and the product B is 6.25 g: 18.75 g: 5-20 mg;
preferably, in the step b-2, the fourth mixed solution and the preliminary reaction product solution are mixed at a volume ratio of 20 mL: 20 mL.
The invention relates to an application of a carboxyl-coated polystyrene AIE dye fluorescence coding microsphere material, which is applied to a liquid-phase chip detection system for protein modification.
Compared with the prior art, the invention has the following obvious and prominent substantive characteristics and remarkable advantages:
1. the invention is different from the design concept of common coding microspheres, the stability of the microspheres is improved by encapsulating and coating the microspheres with a simple carboxyl cross-linked polymer, and the fluorescent coding of the microspheres is realized by a two-step dispersion polymerization method, the synthesis process is simple to operate, the microspheres can keep excellent aggregate fluorescence, and the carboxyl groups on the surfaces can be used for subsequent protein modification;
2. the invention develops a novel coding vector for a liquid phase chip, and provides a novel method for immunoassay, nucleic acid detection analysis and disease diagnosis auxiliary analysis.
Drawings
FIG. 1 is a schematic diagram of the synthetic route for AIE monomers according to a preferred embodiment of the present invention.
FIG. 2 is a schematic diagram of the synthetic route of poly (undecylenic acid) -coated polystyrene AIE dye fluorescent-encoded microspheres according to the preferred embodiment of the present invention.
FIG. 3 is a fluorescent code pattern of microspheres from example 2, example 3, example 4 and example 5 with different concentrations of AIE dye according to a preferred embodiment of the present invention.
Detailed Description
The above-described scheme is further illustrated below with reference to specific embodiments, which are detailed below:
example 1
In this example, referring to FIG. 1, a method of preparing a green AIE dye monomer material comprises the following steps:
a-1, adding 0.3g of zinc powder and 20mL of dry Tetrahydrofuran (THF) into a three-neck flask with the volume of 250mL, slowly dropwise adding 0.36mL of titanium tetrachloride after ice bath, then returning to the room temperature of 20-25 ℃, keeping the temperature for 0.5h, then condensing and refluxing for 2 h, then adding 0.05mL of pyridine, and stirring for at least 10min to obtain a first mixed solution;
a-2, adding 0.2g of 4' -bromobenzophenone and 0.3g of tetraethyl michelia ketone into 20mL of tetrahydrofuran, uniformly stirring to obtain a second mixed solution, slowly and dropwise adding 20mL of the second mixed solution into 20mL of the first mixed solution prepared in the step a-1, and then condensing, refluxing and standing overnight to perform reaction;
a-3, stopping the reaction in the step a-2 by taking 10% by mass of potassium carbonate solution, extracting the product solution by using Dichloromethane (DCM), then performing alkali washing on the collected extract, drying by using anhydrous sodium sulfate, and finally performing column chromatography by using a petroleum ether/ethyl acetate mixed solvent with a volume ratio of 15:1 as an eluent to obtain a product A;
a-4, adding 0.2g of the product A prepared in the step a-3, 0.5g of tetravinyl phenylboronic acid and 20mg of tetrakis (triphenylphosphine) palladium into a three-neck flask with the volume of 100mL, adding 10mL of tetrahydrofuran and 1mL of potassium carbonate solution with the concentration of 1M, refluxing overnight, and reacting to obtain a third mixed solution;
and a-5, extracting the third mixed solution prepared in the step a-4 by using Dichloromethane (DCM), then carrying out alkali washing on the collected extract, drying by using anhydrous sodium sulfate, and finally carrying out column chromatography by using petroleum ether as an eluent to obtain a final product B, namely the green AIE dye monomer material. ,
example 2
In this embodiment, a method for preparing blank polystyrene microspheres by dispersion polymerization includes the following steps:
b-1. dissolving 1g of polyvinylpyrrolidone in 18.75g of absolute ethanol as a solventKetone (PVP), 0.35g Triton X305(Triton X305), 6.25g styrene and 0.25g AIBN were added, the mixed solution was transferred to a 250mL three-necked flask, dispersed by sonication, and N was introduced 2 Keeping the temperature for 30min, and carrying out primary reaction for 1h at 70 ℃;
b-2, adding 6.25g of styrene and 18.75g of absolute ethyl alcohol into a beaker, performing ultrasonic dispersion, and introducing N 2 Keeping the temperature for 30min, preheating at 70 ℃ to obtain a fourth mixed solution, adding 20mL of the obtained fourth mixed solution into a three-necked bottle, mixing with 20mL of the primary reaction product solution in the step b-1, stirring at the rotating speed of 100r/min for 24h, and continuing to react for 24h until the reaction is finished;
b-3, after the reaction in the step b-2 is finished, centrifuging the product solution, and centrifuging the obtained milky white liquid for 3min at the rotating speed of 2000 r/min;
b-4, after centrifugal treatment in the step b-3, ultrasonically cleaning the product for 3 times by using ethanol, and then drying overnight to obtain the polystyrene microsphere.
Example 3
This embodiment is substantially the same as embodiment 2, and is characterized in that:
in this example, referring to fig. 2, a method for preparing polystyrene AIE dye fluorescence-encoded microspheres by dispersion polymerization comprises the following steps:
b-1, dissolving 1g of polyvinylpyrrolidone (PVP) by using 18.75g of absolute ethyl alcohol as a solvent, adding 0.35g of Triton X305(Triton X305), 6.25g of styrene and 0.25g of AIBN, transferring the mixed solution into a 250mL three-neck bottle, ultrasonically dispersing, and introducing N 2 Keeping the temperature for 30min, and carrying out primary reaction for 1h at 70 ℃;
b-2, adding 6.25g of styrene, 18.75g of absolute ethyl alcohol and 5mg of product B into a beaker, performing ultrasonic dispersion, and introducing N 2 Keeping the temperature for 30min, preheating at 70 ℃ to obtain a fourth mixed solution, adding 20mL of the obtained fourth mixed solution into a three-necked bottle, mixing with 20mL of the primary reaction product solution in the step b-1, stirring at the rotating speed of 100r/min for 24h, and continuing to react for 24h until the reaction is finished;
b-3, after the reaction in the step b-2 is finished, centrifuging the product solution, and centrifuging the obtained milky white liquid for 3min at the rotating speed of 2000 r/min;
and b-4, after centrifugal treatment in the step b-3, ultrasonically cleaning the product for 3 times by using ethanol, and then drying overnight to obtain the polystyrene AIE dye fluorescent coding microsphere.
Example 4
This embodiment is substantially the same as the above embodiment, and is characterized in that:
in this example, referring to fig. 2, a method for preparing polystyrene AIE dye fluorescence-encoded microspheres by dispersion polymerization comprises the following steps:
b-1, dissolving 1g of polyvinylpyrrolidone (PVP) by using 18.75g of absolute ethyl alcohol as a solvent, adding 0.35g of Triton X305(Triton X305), 6.25g of styrene and 0.25g of AIBN, transferring the mixed solution into a 250mL three-neck bottle, ultrasonically dispersing, and introducing N 2 Keeping the temperature for 30min, and carrying out primary reaction for 1h at 70 ℃;
b-2, adding 6.25g of styrene, 18.75g of absolute ethyl alcohol and 10mg of product B into a beaker, performing ultrasonic dispersion, and introducing N 2 Keeping the temperature for 30min, preheating at 70 ℃ to obtain a fourth mixed solution, adding 20mL of the obtained fourth mixed solution into a three-necked bottle, mixing with 20mL of the primary reaction product solution in the step b-1, stirring at the rotating speed of 100r/min for 24h, and continuing to react for 24h until the reaction is finished;
b-3, after the reaction in the step b-2 is finished, centrifuging the product solution, and centrifuging the obtained milky white liquid for 3min at the rotating speed of 2000 r/min;
and b-4, after centrifugal treatment in the step b-3, ultrasonically cleaning the product for 3 times by using ethanol, and then drying overnight to obtain the polystyrene AIE dye fluorescent coding microsphere.
Example 5
This embodiment is substantially the same as the above embodiment, and is characterized in that:
in this example, referring to fig. 2, a method for preparing polystyrene AIE dye fluorescence-encoded microspheres by dispersion polymerization comprises the following steps:
b-1, dissolving by using 18.75g of absolute ethyl alcohol as a solvent1g of polyvinylpyrrolidone (PVP), 0.35g of Triton X305(Triton X305), 6.25g of styrene and 0.25g of AIBN were added, the mixed solution was transferred to a 250mL three-necked flask, dispersed by sonication, and N was introduced 2 Keeping the temperature for 30min, and carrying out primary reaction for 1h at 70 ℃;
b-2, adding 6.25g of styrene, 18.75g of absolute ethyl alcohol and 20mg of product B into a beaker, performing ultrasonic dispersion, and introducing N 2 Keeping the temperature for 30min, preheating at 70 ℃ to obtain a fourth mixed solution, adding 20mL of the obtained fourth mixed solution into a three-necked bottle, mixing with 20mL of the primary reaction product solution in the step b-1, stirring at the rotating speed of 100r/min for 24h, and continuing to react for 24h until the reaction is finished;
b-3, after the reaction in the step b-2 is finished, centrifuging the product solution, and centrifuging the obtained milky white liquid for 3min at the rotating speed of 2000 r/min;
and b-4, after centrifugal treatment in the step b-3, ultrasonically cleaning the product for 3 times by using ethanol, and then drying overnight to obtain the polystyrene AIE dye fluorescent coding microsphere.
Example 6
This embodiment is substantially the same as the above embodiment, and is characterized in that:
in this example, referring to FIG. 2, a poly (undecylenic acid) -coated polystyrene AIE dye fluorescently encoded microsphere is prepared by the following steps:
c-1, dispersing 10mg of the polystyrene AIE dye microspheres prepared in the previous embodiment in 10mL of SDS solution with the mass percent of 0.25 wt%, and performing ultrasonic dispersion for 10min to obtain a polystyrene AIE dye microsphere dispersion solution;
c-2, sequentially adding 12.5mg of KPS, a mixed solution of DVB and methanol with a volume ratio of 60uL being 1:100 and a mixed solution of undecylenic acid and methanol with a volume ratio of 100uL being 1:100 into the polystyrene AIE dye microsphere dispersion liquid prepared in the step c-1, and performing ultrasonic dispersion on the mixed solution for 10min to obtain a precursor mixed solution;
and c-3, placing the precursor mixed solution prepared in the step c-2 into a three-neck flask with the volume of 25mL, stirring for 10min, gradually heating to 70 ℃, reacting for 8 hours, then centrifugally cleaning the product solution with water for three times, and then cleaning the product with ethanol for three times to finally obtain the polystyrene AIE dye fluorescent coding microspheres with outer layers coated with carboxyl.
Example 7
This embodiment is substantially the same as the above embodiment, and is characterized in that:
in this example, 10mg of the carboxyl-coated polystyrene AIE dye fluorescence-encoded microspheres prepared in example 2, example 3, example 4 and example 5 were dispersed in 2mL of aqueous solution and ultrasonically dispersed for 10min to obtain experimental samples corresponding to the carboxyl-coated polystyrene AIE dye fluorescence-encoded microspheres prepared in example 2, example 3, example 4 and example 5, respectively, and the following experimental test analyses were respectively performed:
10uL of each sample was added to the flow tube for fluorescence flow detection, and the results are shown in FIG. 3. FIG. 3 is a fluorescent code pattern of microspheres from example 2, example 3, example 4 and example 5 using different concentrations of AIE dye in this example. As can be seen in fig. 3, the KO525 fluorescence channel of each sample exhibited fluorescence with different characteristic differences. As can be seen from FIG. 3 in conjunction with this example, microspheres without added fluorescent AIE monomer, with fluorescence peaks detected by flow at only 2 x 10 < SP > 2 </SP > intensity as shown in example 2, while microspheres with added AIE fluorescent monomers of 5mg,10mg,20mg, with fluorescence peaks detected by flow at 5 x 10 < SP > 4 </SP >, 1 x 10 < SP > 5 </SP >, 2 < SP > 10 < SP > 5 </SP > respectively as shown in examples 3, 4, 5, produced a clear contrast to example 2, and the fluorescence peaks between examples 3, 4, 5 were relatively open with essentially no overlap, thereby successfully achieving microsphere fluorescence encoding.
In the embodiment of the invention, the microspheres and the AIE material are combined, the microspheres with the average diameter of 3 mu m are prepared by a two-step dispersion polymerization method, and functional groups such as carboxyl and the like are modified on the surfaces of the microspheres. The invention designs the polymer microsphere of the novel poly AIE fluorescent dye, ensures the stability of the microsphere, simultaneously realizes fluorescent coding by adjusting the amount of the fluorescent dye in the polymerization process, and can be more conveniently used for protein modification because the polymer surface contains a large amount of carboxyl.
In a word, the invention combines two sections of dispersion polymerization, aggregation induced emission, AIEgen and fluorescence encoding to prepare the monodisperse polystyrene microsphere with the aggregation fluorescence effect (AIE), and the microsphere has wide application prospect in the aspect of fluorescence encoding. Fluorescent AIE monomers were first synthesized by Suzuki reaction and confirmed by Nuclear Magnetic Resonance (NMR). These AIE monomers (AIEgens) exhibit bright green fluorescence in the solid state, while exhibiting increased fluorescence with increasing water content in aqueous Tetrahydrofuran (THF). The invention adopts a two-step dispersion polymerization method to prepare micron-sized monodisperse polystyrene microspheres of copolymerized AIE molecules, and then the micron-sized monodisperse polystyrene microspheres are subjected to carboxylation modification to obtain functional polystyrene microspheres with CV of less than 3%. The undecylenic acid endows a large number of carboxyl groups of the coded microspheres with convenience for protein modification and further application to a liquid-phase chip detection system. Therefore, the novel green AIE fluorescent coding microspheres can be applied to various biomedical industries such as liquid phase chips.
The embodiments of the present invention have been described with reference to the accompanying drawings, but the present invention is not limited to the embodiments, and various changes and modifications can be made according to the purpose of the invention, and any changes, modifications, substitutions, combinations or simplifications made according to the spirit and principle of the technical solution of the present invention shall be equivalent substitutions, as long as the purpose of the present invention is met, and the present invention shall fall within the protection scope of the present invention without departing from the technical principle and inventive concept of the present invention.
Claims (10)
1. A polystyrene AIE dye fluorescence coding microsphere material with an outer layer coated with carboxyl is characterized in that: preparing an AIE monomer by a two-step synthesis method, then carrying out two-step dispersion polymerization reaction on the AIE monomer, and coating undecylenic acid on the surface of a polymerization product, thereby modifying carboxyl functional groups on the surface of the microsphere and finally obtaining the polystyrene AIE dye fluorescence coding microsphere material containing carboxyl on the surface.
2. The carboxyl-coated polystyrene AIE dye fluorescent coding microsphere material of claim 1, which is characterized in that: the polystyrene AIE dye fluorescent coding microsphere is a microsphere with the average grain diameter of 3 +/-0.1 mu m.
3. A method for preparing the carboxyl-coated polystyrene AIE dye fluorescent coding microsphere material of claim 1, which is characterized by comprising the following steps:
a. preparation of green AIE dye monomer material:
a-1, adding zinc powder and 20mL of dry Tetrahydrofuran (THF) into a three-neck flask with the volume of 250mL, slowly dropwise adding titanium tetrachloride after ice bath, then returning to room temperature, keeping for at least 0.5h, carrying out condensation reflux for at least 2 hours, then adding pyridine, and stirring for at least 10 minutes to obtain a first mixed solution;
a-2, adding 4' -bromobenzophenone and tetraethyl michigan ketone into 20mL of tetrahydrofuran, uniformly stirring to obtain a second mixed solution, slowly dropwise adding the second mixed solution into the first mixed solution prepared in the step a-1, and then condensing, refluxing and standing overnight for reaction;
a-3, stopping the reaction in the step a-2 by taking a potassium carbonate solution with the mass percent not less than 10%, extracting a product solution by using Dichloromethane (DCM), then performing alkali washing on the collected extract, drying by using anhydrous sodium sulfate, and finally performing column chromatography by using a petroleum ether/ethyl acetate mixed solvent with the volume ratio of 15:1 as an eluent to obtain a product A;
a-4, adding the product A prepared in the step a-3, the tetravinylbenzene boric acid and the tetrakis (triphenylphosphine) palladium into a three-neck flask with the volume of 100mL, adding 10mL of tetrahydrofuran and a potassium carbonate solution with the mass percent not less than 10%, refluxing overnight, and reacting to obtain a third mixed solution;
a-5, extracting the third mixed solution prepared in the step a-4 by using Dichloromethane (DCM), then performing alkali washing on the collected extract, drying by using anhydrous sodium sulfate, and finally performing column chromatography by using petroleum ether as an eluent to obtain a final product B, namely the green AIE dye monomer material;
b. preparing the polystyrene AIE dye fluorescent coding microspheres by dispersion polymerization:
b-1, dissolving polyvinylpyrrolidone (PVP) by using absolute ethyl alcohol as a solvent, adding Triton X305(Triton X305), styrene and AIBN, transferring the mixed solution into a 250mL three-neck bottle, performing ultrasonic dispersion, and introducing N 2 At least 30min, and carrying out primary reaction at the temperature of not less than 70 ℃ for at least 1 h;
b-2, adding styrene, absolute ethyl alcohol and 5-20mg of product B into a beaker, performing ultrasonic dispersion, and introducing N 2 Preheating at the temperature of not less than 70 ℃ for at least 30min to obtain a fourth mixed solution, adding the obtained fourth mixed solution into a three-necked bottle, mixing the fourth mixed solution with the primary reaction product solution in the step b-1, and continuously reacting for at least 24h until the reaction is finished;
b-3, after the reaction in the step b-2 is finished, centrifuging the product solution, and centrifuging the obtained milky white liquid for at least 3min at a rotating speed of not less than 2000 r/min;
b-4, after centrifugal treatment in the step b-3, ultrasonically cleaning the product for at least 3 times by using ethanol, and then drying overnight to obtain the polystyrene AIE dye fluorescent coding microsphere;
c. preparing poly (undecylenic acid) coated polystyrene AIE dye fluorescent coding microspheres:
c-1, dispersing 10mg of polystyrene AIE dye microspheres in 10mL of SDS solution with the mass percent of not less than 0.25 wt%, and performing ultrasonic dispersion for at least 10min to obtain a polystyrene AIE dye microsphere dispersion solution;
c-2, sequentially adding 12.5mg KPS, a mixed solution of DVB and methanol with a volume ratio of 60uL being 1:100 and a methanol solution of undecylenic acid with a volume ratio of 100uL being 1:100 into the polystyrene AIE dye microsphere dispersion liquid prepared in the step c-1, and performing ultrasonic dispersion on the mixed solution for at least 10min to obtain a precursor mixed solution;
and c-3, placing the precursor mixed solution prepared in the step c-2 into a three-necked bottle with the volume of 25mL, stirring for at least 10min, gradually heating to a temperature of not less than 70 ℃, reacting for at least 8 hours, then centrifugally cleaning the product solution with water for at least three times, and cleaning the product with ethanol for at least three times to finally obtain the polystyrene AIE dye fluorescence encoding microsphere with the carboxyl coated on the outer layer.
4. The preparation method of the outer-layer carboxyl-coated polystyrene AIE dye fluorescent coding microsphere material according to claim 3, which is characterized in that: in the step a-1, the mixing ratio of zinc powder, tetrahydrofuran, titanium tetrachloride and pyridine is 0.3 g: 20mL of: 0.36 ml: 0.05 mL.
5. The preparation method of the outer-layer carboxyl-coated polystyrene AIE dye fluorescent coding microsphere material according to claim 3, which is characterized in that: in the step a-2, the mixing ratio of tetrahydrofuran, 4' -bromobenzophenone and tetraethyl michelson is 20 mL: 0.2 g: 0.3 g.
6. The preparation method of the outer-layer carboxyl-coated polystyrene AIE dye fluorescent coding microsphere material according to claim 3, which is characterized in that: in the step a-2, the volume ratio of the second mixed solution to the first mixed solution is 20 mL: 20 mL.
7. The preparation method of the outer-layer carboxyl-coated polystyrene AIE dye fluorescent coding microsphere material according to claim 3, which is characterized in that: in said step a-4, the product A, tetravinylbenzeneboronic acid and tetrakis (triphenylphosphine) palladium were mixed in a ratio of 0.2 g: 0.5 g: 20 mg; the adding amount of the potassium carbonate solution with the mass percent not less than 10 percent is not less than 1 mL.
8. The preparation method of the outer-layer carboxyl-coated polystyrene AIE dye fluorescent coding microsphere material according to claim 3, which is characterized in that: in the step b-1, the mass ratio of the absolute ethyl alcohol to the polyvinylpyrrolidone to the triton X305 to the styrene to the AIBN is 18.75:1:0.35:6.25: 0.25.
9. The preparation method of the outer-layer carboxyl-coated polystyrene AIE dye fluorescent coding microsphere material according to claim 3, which is characterized in that: in the step B-2, the mixing ratio of the styrene, the absolute ethyl alcohol and the product B is 6.25 g: 18.75 g: 5-20 mg;
alternatively, in the step b-2, the fourth mixed solution and the preliminary reaction product solution are mixed at a volume ratio of 20 mL: 20 mL.
10. The application of the carboxyl-coated polystyrene AIE dye fluorescent coding microsphere material disclosed by claim 1, which is characterized in that the carboxyl-coated polystyrene AIE dye fluorescent coding microsphere material is applied to a liquid-phase chip detection system for protein modification.
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